Transcriptional programming of cell identity is gaining importance at both basic developmental and clinical level. While the phenomenology of cell programming and reprogramming by forced expression of transcription factors is well described, the mechanism of action of programming factors or the sequence of regulatory events resulting in a cell adopting a new identity are largely unknown. We propose to combine the strengths of stem cell biology with genomic and computational approaches to characterize the process of transcriptional programming of motor neuron (MN) identity. We developed efficient methods for the induction of MN identity in differentiating embryonic stem cells (ESCs) by the expression of defined transcription factors. Using the system we will combine biochemical, genomic and computational analysis to address following questions: i) whether programming factors directly regulate terminal motor neuron effector genes or initiate a cascade of intermediate transcription programs; ii) whether recruitment of programming factors to DNA is cooperative and which factors determine the specificity of DNA binding; iii) whether identified MN enhancers are inaccessible for programming factor binding in cell types refractory to MN programming; iv) whether identified secondary binding motifs for Onecut and Ebf transcription factors contribute to productive regulation of MN specific gene expression; v) whether additional secondary motifs recruit ancillary transcription factors to NIL bound enhancers that contribute to productive regulation of MN specific gene expression. Together these studies will provide fundamental insight into the developmental processes underlying specification of defined cell identity and will provide novel and efficient source of motor neurons for disease modeling, study and drug discovery.